Mycobacteria are important intracellular human pathogens. Tuberculosis is the leading cause of death in the world secondary to an infectious disease. The ability of M. tuberculosis to cause widespread infection depends on the organism's ability to escape host defenses by surviving, replicating, and escaping from the macrophage. There are two major goals of this research proposal. The first main objective is to identify and characterize mycobacterial genes expressed specifically within macrophages which are responsible for intracellular replication and survival. To identify macrophage-specific promoters from mycobacteria, a selective assay using Thy- mutants to screen for these genes intracellularly will be developed. An auxotrophic mutant of mycobacteria is required for this assay. We have successfully cloned the thymidylate synthase gene from an M. smegmatis genomic library in E. coli and are currently modifying this gene to create the auxotrophic mutant Thy-in M. smegmatis and in BCG using recombinant technology. Mycobacterial mutants which are unable to grow within macrophages will be identified by using an intracellular thymineless death enrichment strategy which has been used successfully in L. pneumophila. The second objective is to develop a general approach to identify secreted gene products from M. tuberculosis which may play an important role in its pathogenesis. The construction of an M. tuberculosis phoA expression library has yielded a lipoprotein from an initial screen. This lipoprotein will be further characterized. This phoA expression library may prove to be a powerful tool to selectively identify M. tuberculosis secreted proteins which may be involved in its transport mechanisms, pathogenicity, and antigenicity Progression towards these goals may yield new genetic and molecular biologic techniques to study mycobacteria and may contribute to the fundamental understanding of mycobacterial pathogenesis and its virulence. The identification of these genes may be potential targets for new antibiotics and an attenuated strain may serve as a live vaccine.